RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods

Abstract

RNA enzymes or ribozymes can act as endoribonucleases, catalyzing the cleavage of RNA molecules with a sequence specificity of cleavage greater than that of known ribonucleases and approaching that of the DNA restriction endonucleases, thus serving as RNA sequence specific endoribonucleases. An example is a shortened form of the self-splicing ribosomal RNA intervening sequence of Tetrahymena (L-19 IVS RNA). Site-specific mutagenesis of the enzyme active site of the L-19 IVS RNA alters the substrate sequence specificity in a predictable manner, allowing a set of sequence-specific endoribonucleases to be synthesized. Varying conditions allow the ribozyme to act as a polymerase (nucleotidyltransferase), a dephosphorylase (acid phosphatase or phosphotransferase) or a sequence-specific endoribonuclease.

What is claimed:  
     
       1. A method comprising the steps of: 
       a. contacting an RNA molecule having endonuclease activity with a sample under conditions suitable for the RNA molecule having endonuclease activity to catalyze an endonuclease reaction on a separate RNA molecule wherein said endonuclease reaction is a transesterification reaction; and  
       b. assaying for the endonuclease reaction catalyzed by the RNA molecule having endonuclease activity in step (a).  
     
     
       2. The method of  claim 1 , wherein said separate RNA molecule is an RNA molecule comprising at least about 145 nucleotides. 
     
     
       3. The method of  claim 2 , wherein said large RNA molecule is a pre-mRNA molecule. 
     
     
       4. The method of  claim 1 , wherein more than one RNA molecule having endonuclease activity is contacted with a sample in step (a). 
     
     
       5. The method of  claim 1 , wherein said method is an in vitro method. 
     
     
       6. The method of  claim 1 , wherein said sample is an RNA sample. 
     
     
       7. The method of  claim 1 , wherein said RNA molecule having endonuclease activity comprises ribonucleotides. 
     
     
       8. The method of  claim 1 , wherein said RNA molecule having endonuclease activity is chemically synthesized. 
     
     
       9. The method of  claim 1 , wherein said RNA molecule having endonuclease activity is enzymatically synthesized.